C peptide detection by mass spectrometry

ABSTRACT

Methods are described for measuring the amount of C peptide in a sample. More specifically, mass spectrometric methods are described for detecting and quantifying C peptide in a sample utilizing on-line extraction methods coupled with tandem mass spectrometric or high resolution/high accuracy mass spectrometric techniques.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/827,333, filed May 27, 2022, which is a continuation of U.S.application Ser. No. 17/027,267, filed Sep. 21, 2020, now U.S. Pat. No.11,346,845, which is a continuation of U.S. application Ser. No.16/430,030, filed Jun. 3, 2019, now U.S. Pat. No. 10,782,305, which is acontinuation of U.S. application Ser. No. 16/259,606, filed Jan. 28,2019, now U.S. Pat. No. 10,309,972, which is a continuation of U.S.application Ser. No. 15/939,030, filed Mar. 28, 2018, now U.S. Pat. No.10,191,065, which is a continuation of U.S. application Ser. No.15/888,681, filed Feb. 5, 2018, now U.S. Pat. No. 9,964,546, which is acontinuation of U.S. application Ser. No. 15/457,683, filed Mar. 13,2017, now U.S. Pat. No. 9,885,724, which is a continuation of U.S.application Ser. No. 14/654,964, filed Jun. 23, 2015, now U.S. Pat. No.9,594,074, which is a national stage application of InternationalApplication No. PCT/US2013/077575, filed Dec. 23, 2013, which claims thebenefit under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser.No. 61/745,976, filed Dec. 26, 2012, the contents of which areincorporated by reference in their entirety into the present disclosure.

FIELD OF THE INVENTION

The invention relates to the quantitative measurement of C peptide. In aparticular aspect, the invention relates to methods for quantitativemeasurement of C peptide by mass spectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

C peptide is a peptide that is formed as part of the process ofproinsulin conversion (via cleavage) to insulin before release fromendocytic vesicles within the pancreas. Human C peptide has a molar massof about 3020.3 amu.

C peptide binds to receptors at the cell surface and activates signaltransduction pathways that result in stimulation of Na+, K+ ATPase andendothelial nitric oxide synthase (eNOS). Both of these enzymes havereduced activities in type 2 diabetes. C peptide also functions inrepair of the muscular layer of the arteries.

C peptide levels instead of insulin are often measured in newlydiagnosed diabetes patients because insulin concentration in the portalvein can range from two to ten times higher than in the peripheralcirculation. The liver extracts about half of the insulin from plasma,but this varies with the nutritional state of the subject. Thus, Cpeptide may be a more comprehensive indicator of insulin status thandirect insulin measurement. Patients with type 1 diabetes are unable toproduce insulin efficiently and therefore will have a decreased level ofC peptide, while C peptide levels in patients with type 2 diabetes aretypically normal or even elevated. Thus, C peptide measurement is usedto distinguish type 1 diabetes from type 2 diabetes. Additionally, as Cpeptide is formed during natural insulin production, measuring C peptidein patients undergoing insulin therapy may help determine how muchnatural insulin the patient is producing.

C peptide measurement can also be used to determine if a patient mayhave a gastrinoma associated with Multiple Endocrine Neoplasm syndrome.A significant number of Multiple Endocrine Neoplasm syndromes presentingwith gastrinoma also include pancreatic, parathyroid, and pituitaryadenomas. Higher levels of C peptide together with the presence of agastrinoma suggests that organs other than the stomach may harbor aneoplasm. C peptide may also be assessed in patients suspected ofinsulin abuse, and in women with Polycystic Ovary Syndrome to assessdegree of insulin resistance.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the presence oramount of C peptide in a sample by mass spectrometry.

Some embodiments presented herein utilize tandem mass spectrometry. Insome of these embodiments, the methods include: (a) subjecting a samplesuspected of containing C peptide to high performance liquidchromatography (HPLC) to obtain a fraction enriched in C peptide; (b)subjecting the enriched C peptide to an ionization source underconditions suitable to generate one or more C peptide ions detectable bymass spectrometry; (c) determining the amount of one or more C peptideions by tandem mass spectrometry, wherein the determined ions comprise aprecursor ion with a mass to charge ratio of 1007.5±0.5 and one or morefragment ions selected from the group of ions with mass to charge ratiosconsisting of 927.6±0.5, 785.4±0.5, and 646.1±0.5. In these embodiments,the amount of the one or more ions determined in step (c) is related tothe amount of C peptide in the sample, e.g., used to determine theamount of C peptide in the sample. In some embodiments, the HPLC is 1-DHPLC. In some embodiments, the amounts of two or more fragment ionsselected from the group consisting of 927.6±0.5, 785.4±0.5, and646.1±0.5 are determined in step (c).

In other embodiments utilizing tandem mass spectrometry, the methodsinclude: (a) subjecting the sample to 1-D high performance liquidchromatography (1-D HPLC) to obtain a fraction enriched in C peptide;(b) subjecting the fraction enriched in C peptide to an ionizationsource under conditions suitable to generate one or more C peptide ionsdetectable by mass spectrometry; and (c) determining the amount of oneor more C peptide ions by tandem mass spectrometry. In theseembodiments, the amount of ions determined in step (c) is related to theamount of a C peptide in the sample. In some embodiments, the one ormore ions detected in step (c) comprise a precursor ion with a mass tocharge ratio (m/z) of about 1007.5±0.5. In some related embodiments, theone or more ions detected in step (c) further comprise one or morefragment ions selected from the group of ions with mass to charge ratios(m/z) of about 927.6±0.5, 785.4±0.5, and 646.1±0.5. In some relatedembodiments, the one or more ions detected in step (c) comprise two ormore fragment ions selected from the group of ions with mass to chargeratios (m/z) of about 927.6±0.5, 785.4±0.5, and 646.1±0.5.

In embodiments utilizing tandem mass spectrometry, tandem massspectrometry may be conducted by any method known in the art, includingfor example, multiple reaction monitoring, precursor ion scanning, orproduct ion scanning.

In some embodiments, tandem mass spectrometry comprises fragmenting aprecursor ion with a mass to charge ratio of 1007.5±0.50 into one ormore fragment ions. In certain related embodiments, the one or morefragment ions comprise one or more ions selected from the groupconsisting of ions with mass to charge ratios of 927.6±0.5,785.4±646.1±0.5, 147.0±0.5, and 260.3±0.5. In other related embodiments,the one or more fragment ions comprise one or more ions selected fromthe group consisting of ions with mass to charge ratios of 927.6±0.5,785.4±0.5, 646.1±0.5. In embodiments where the amounts of two or morefragment ions are determined, the amounts may be subject to anymathematical manipulation known in the art in order to relate themeasured ion amounts to the amount of C peptide in the sample. Forexample, the amounts of two or more fragment ions may be summed as partof determining the amount of C peptide in the sample.

Some embodiments presented herein utilizing high resolution/highaccuracy mass spectrometry. In some of these embodiments, the methodsinclude: (a) subjecting a sample suspected of containing C peptide tohigh performance liquid chromatography (HPLC) to obtain a fractionenriched in C peptide; (b) subjecting the fraction enriched in C peptideto an ionization source under conditions suitable to generate one ormore C peptide ions detectable by mass spectrometry; and (c) determiningthe amount of one or more C peptide ions by high resolution/highaccuracy mass spectrometry. In these embodiments, the amount of ionsdetermined in step (c) is related to the amount of C peptide in thesample.

In some embodiments, high resolution/high accuracy spectrometry isconducted at a resolving power or FWHM (Full Width at Half Maximum) of10,000 and a mass accuracy of 50 ppm. In some embodiments, the highresolution/high accuracy mass spectrometer is a high resolution/highaccuracy time-of-flight (TOF) mass spectrometer. In some embodiments,HPLC is 1-D HPLC. In some embodiments, the one or more ions determinedin step (c) comprise an ion with a charge of 2+ or 3+. In someembodiments, the one or more ions determined in step (c) comprise an ionselected from the group of ions with a mass to charge ratio (m/z) withinthe ranges of about 1007.5±1 and 1510.3±1.

In some embodiments, the high resolution/high accuracy mass spectrometryis conducted at a resolving power (FWHM) of greater than or equal toabout 10,000, such as greater than or equal to about 15,000, such asgreater than or equal to about 20,000, such as greater than or equal toabout 25,000. In some embodiments, the high resolution/high accuracymass spectrometry is conducted at an accuracy of less than or equal toabout 50 ppm, such as less than or equal to about 20 ppm, such as lessthan or equal to about 10 ppm, such as less than or equal to about 5ppm; such as less than or equal to about 3 ppm. In some embodiments,high resolution/high accuracy mass spectrometry is conducted at aresolving power (FWHM) of greater than or equal to about 10,000 and anaccuracy of less than or equal to about 50 ppm. In some embodiments, theresolving power is greater than about 15,000 and the accuracy is lessthan or equal to about 20 ppm. In some embodiments, the resolving poweris greater than or equal to about 20,000 and the accuracy is less thanor equal to about 10 ppm; preferably resolving power is greater than orequal to about 20,000 and accuracy is less than or equal to about 5 ppm,such as less than or equal to about 3 ppm.

In some embodiments, the high resolution/high accuracy mass spectrometrymay be conducted with an orbitrap mass spectrometer, a time of flight(TOF) mass spectrometer, or a Fourier transform ion cyclotron resonancemass spectrometer (sometimes known as a Fourier transform massspectrometer).

In any of the methods described herein, the sample may comprise abiological sample. In some embodiments, the biological sample maycomprise a body fluid such as urine, plasma, or serum. In someembodiments, the biological sample may comprise a sample from a human;such as from an adult male or female, or juvenile male or female,wherein the juvenile is under age 18, under age 15, under age 12, orunder age 10. The human sample may be analyzed to diagnose or monitor adisease state or condition, or to monitor therapeutic efficacy oftreatment of a disease state or condition. In some related embodiments,the methods described herein may be used to determine the amount of Cpeptide in a biological sample when taken from a human.

In embodiments utilizing either tandem mass spectrometry or highresolution/high accuracy mass spectrometry, the sample may be subjectedto high performance liquid chromatography (HPLC) prior to ionization.

In embodiments utilizing either tandem mass spectrometry or highresolution/high accuracy mass spectrometry, the sample may be subjectedto an extraction column, such as a solid phase extraction (SPE) column,prior to being subjected to an analytical column, such as a highperformance liquid chromatography (HPLC) column. In some relatedembodiments, the extraction column is not an immunopurification column(i.e., an immunoaffinity column). In some embodiments,immunopurification is not used at any point in the method. In alternateembodiments, C peptide is extracted from the sample with animmunopurification technique; such as with an immunoaffinity extractioncolumn.

In embodiments which utilize two or more of an extraction column such asa solid phase extraction column (SPE), an analytical column such as ahigh performance liquid chromatography (HPLC) column, and an ionizationsource, two or more of these components may be connected in an on-linefashion to allow for automated sample processing and analysis.

In any of the methods presented herein, the sample may comprise abiological sample; such as a body fluid sample, including, for example,plasma or serum.

Mass spectrometry (either tandem or high resolution/high accuracy) maybe performed in positive ion mode. Alternatively, mass spectrometry maybe performed in negative ion mode. Various ionization sources, includingfor example atmospheric pressure chemical ionization (APCI) orelectrospray ionization (ESI), may be used to ionize C peptide. In someembodiments, C peptide is ionized by ESI in positive ion mode.

In any method presented herein, a separately detectable internalstandard may be provided in the sample, the amount of which is alsodetermined in the sample. In embodiments utilizing a separatelydetectable internal standard, all or a portion of both the analyte ofinterest and the internal standard present in the sample is ionized toproduce a plurality of ions detectable in a mass spectrometer, and oneor more ions produced from each are detected by mass spectrometry. Inthese embodiments, the presence or amount of ions generated from theanalyte of interest may be related to the presence of amount of analyteof interest in the sample by comparison to the amount of internalstandard ions detected.

Alternatively, the amount of the C peptide in a sample may be determinedby comparison to one or more external reference standards. Exemplaryexternal reference standards include blank plasma or serum spiked with Cpeptide or an isotopically labeled variant thereof.

In some embodiments, the methods demonstrate a linear range fordetection of C peptide at levels at least within the range of about0.049 ng/50 μL to 25 ng/50 μL.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the terms “purification”, “purifying”, and “enriching”do not refer to removing all materials from the sample other than theanalyte(s) of interest. Instead, these terms refer to a procedure thatenriches the amount of one or more analytes of interest relative toother components in the sample that may interfere with detection of theanalyte of interest. Purification of the sample by various means mayallow relative reduction of one or more interfering substances, e.g.,one or more substances that may or may not interfere with the detectionof selected parent or daughter ions by mass spectrometry. Relativereduction as this term is used does not require that any substance,present with the analyte of interest in the material to be purified, isentirely removed by purification.

As used herein, the term “immunopurification” or “immunopurify” refersto a purification procedure that utilizes antibodies, includingpolyclonal or monoclonal antibodies, to enrich the one or more analytesof interest. Immunopurification can be performed using any of theimmunopurification methods well known in the art. Often theimmunopurification procedure utilizes antibodies bound, conjugated orotherwise attached to a solid support, for example a column, well, tube,gel, capsule, particle or the like. Immunopurification as used hereinincludes without limitation procedures often referred to in the art asimmunoprecipitation, as well as procedures often referred to in the artas affinity chromatography or immunoaffinity chromatography.

As used herein, the term “immunoparticle” refers to a capsule, bead, gelparticle or the like that has antibodies bound, conjugated or otherwiseattached to its surface (either on and/or in the particle). In certainpreferred embodiments, immunoparticles are sepharose or agarose beads.In alternative preferred embodiments, immunoparticles comprise glass,plastic or silica beads, or silica gel.

As used herein, the term “anti-C peptide antibody” refers to anypolyclonal or monoclonal antibody that has an affinity for C peptide. Invarious embodiments the specificity of C peptide antibodies to chemicalspecies other than C peptide may vary; for example in certain preferredembodiments the anti-C peptide antibodies are specific for C peptide andthus have little or no affinity for chemical species other than Cpeptide, whereas in other preferred embodiments the anti-C peptideantibodies are non-specific and thus bind certain chemical species otherthan C peptide.

As used herein, the term “sample” refers to any sample that may containan analyte of interest. As used herein, the term “body fluid” means anyfluid that can be isolated from the body of an individual. For example,“body fluid” may include blood, plasma, serum, bile, saliva, urine,tears, perspiration, and the like. In certain embodiments, the samplecomprises a body fluid sample from a human; such as plasma or serum.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis. SPE, including TFLC, may operate via a unitary or mixed modemechanism. Mixed mode mechanisms utilize ion exchange and hydrophobicretention in the same column; for example, the solid phase of amixed-mode SPE column may exhibit strong anion exchange and hydrophobicretention; or may exhibit column exhibit strong cation exchange andhydrophobic retention.

Generally, the affinity of a SPE column packing material for an analytemay be due to any of a variety of mechanisms, such as one or morechemical interactions or an immunoaffinity interaction. In someembodiments, SPE of C peptide is conducted without the use of animmunoaffinity column packing material. That is, in some embodiments,insulin is purified from a sample by a SPE column that is not animmunoaffinity column.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column. The term “1-D high performance liquidchromatography” or “1-D HPLC” refers to traditional, single column HPLC.The term “2-D high performance liquid chromatography” refers to a highperformance liquid chromatography technique where two HPLC columns areused in such a way that the analyte and any additional species thatco-elute at the same time as the analyte are directed from a first HPLCcolumn onto a second HPLC column with a different stationary phase. Thestationary phase of the second HPLC column is selected such that theanalyte and co-eluting species are separated before introduction of theanalyte to a mass spectrometric instrument. 2-D HPLC typically is morecostly in terms of run time and requires additional complexity of set-uprelative to 1-D HPLC; however, in particularly complex samples, greateranalyte purity may be achieved with 2-D HPLC compared to 1-D HPLC.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow”. When fluid flowsslowly and smoothly, the flow is called “laminar flow”. For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in substantially straight lines. At fastervelocities, the inertia of the water overcomes fluid frictional forcesand turbulent flow results. Fluid not in contact with the irregularboundary “outruns” that which is slowed by friction or deflected by anuneven surface. When a fluid is flowing turbulently, it flows in eddiesand whirls (or vortices), with more “drag” than when the flow islaminar. Many references are available for assisting in determining whenfluid flow is laminar or turbulent (e.g., “Turbulent Flow Analysis:Measurement and Prediction,” P. S. Bernard & J. M. Wallace, John Wiley &Sons, Inc., (2000); “An Introduction to Turbulent Flow,” Jean Mathieu &Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm. As used in this context, the term“about” means ±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means ±10%. In apreferred embodiment the analytical column contains particles of about 5μm in diameter.

As used herein, the terms “on-line” and “inline”, for example as used in“on-line automated fashion” or “on-line extraction”, refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation and the supernatants are then manually loadedinto an autosampler, the precipitation and loading steps are off-linefrom the subsequent steps. In various embodiments of the methods, one ormore steps may be performed in an on-line automated fashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer, amass analyzer, and an ion detector. In general, one or more molecules ofinterest are ionized, and the ions are subsequently introduced into amass spectrometric instrument where, due to a combination of magneticand electric fields, the ions follow a path in space that is dependentupon mass (“m”) and charge (“z”). See, e.g., U.S. Pat. Nos. 6,204,500,entitled “Mass Spectrometry From Surfaces;” 6,107,623, entitled “Methodsand Apparatus for Tandem Mass Spectrometry;” 6,268,144, entitled “DNADiagnostics Based On Mass Spectrometry;” 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis2000, 21: 1164-67.

As used herein, “high resolution/high accuracy mass spectrometry” refersto mass spectrometry conducted with a mass analyzer capable of measuringthe mass to charge ratio of a charged species with sufficient precisionand accuracy to confirm a unique chemical ion. Confirmation of a uniquechemical ion is possible for an ion when individual isotopic peaks fromthat ion are readily discernable. The particular resolving power andmass accuracy necessary to confirm a unique chemical ion varies with themass and charge state of the ion.

As used herein, the term “resolving power” or “resolving power (FWHM)”(also known in the art as “m/Δm_(50%)”) refers to an observed mass tocharge ratio divided by the width of the mass peak at 50% maximum height(Full Width Half Maximum, “FWHM”). The effect of differences inresolving power is illustrated, for example, in FIGS. 1A-C of co-pendingU.S. Patent Publication No. 2011/0111512, which is incorporated byreference herein.

As used herein a “unique chemical ion” with respect to mass spectrometryrefers a single ion with a single atomic makeup. The single ion may besingly or multiply charged.

As used herein, the term “accuracy” (or “mass accuracy”) with respect tomass spectrometry refers to potential deviation of the instrumentresponse from the true m/z of the ion investigated. Accuracy istypically expressed in parts per million (ppm). The effect ofdifferences in mass accuracy is illustrated, for example, in FIGS. 2A-Dof co-pending U.S. Patent Publication No. 2011/0111512, which isincorporated by reference herein.

High resolution/high accuracy mass spectrometry methods of the presentinvention may be conducted on instruments capable of performing massanalysis with FWHM of greater than 10,000, 15,000, 20,000, 25,000,50,000, 100,000, or even more. Likewise, methods of the presentinvention may be conducted on instruments capable of performing massanalysis with accuracy of less than 50 ppm, 20 ppm, 15 ppm, 10 ppm, 5ppm, 3 ppm, or even less. Instruments capable of these performancecharacteristics may incorporate certain orbitrap mass analyzers,time-of-flight (“TOF”) mass analyzers, or Fourier-transform ioncyclotron resonance mass analyzers. In preferred embodiments, themethods are carried out with an instrument which includes an orbitrapmass analyzer or a TOF mass analyzer.

The term “orbitrap” describes an ion trap consisting of an outerbarrel-like electrode and a coaxial inner electrode. Ions are injectedtangentially into the electric field between the electrodes and trappedbecause electrostatic interactions between the ions and electrodes arebalanced by centrifugal forces as the ions orbit the coaxial innerelectrode. As an ion orbits the coaxial inner electrode, the orbitalpath of a trapped ion oscillates along the axis of the central electrodeat a harmonic frequency relative to the mass to charge ratio of the ion.Detection of the orbital oscillation frequency allows the orbitrap to beused as a mass analyzer with high accuracy (as low as 1-2 ppm) and highresolving power (FWHM) (up to about 200,000). A mass analyzer based onan orbitrap is described in detail in U.S. Pat. No. 6,995,364,incorporated by reference herein in its entirety. Use of orbitrapanalyzers has been reported for qualitative and quantitative analyses ofvarious analytes. See, e.g., U.S. Patent Application Pub. No.2008/0118932 (filed Nov. 9, 2007); Bredehöft, et al., Rapid Commun. MassSpectrom., 2008, 22:477-485; Le Breton, et al., Rapid Commun. MassSpectrom., 2008, 22:3130-36; Thevis, et al., Mass Spectrom. Reviews,2008, 27:35-50; Thomas, et al., J. Mass Spectrom., 2008, 43:908-15;Schenk, et al., BMC Medical Genomics, 2008, 1:41; and Olsen, et al.,Nature Methods, 2007, 4:709-12.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected. In preferred embodiments, mass spectrometry isconducted in positive ion mode.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber. As the droplets get smaller the electrical surface chargedensity increases until such time that the natural repulsion betweenlike charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for theionization of molecule M is photon absorption and electron ejection toform the molecular ion M+. Because the photon energy typically is justabove the ionization potential, the molecular ion is less susceptible todissociation. In many cases it may be possible to analyze sampleswithout the need for chromatography, thus saving significant time andexpense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser desorption thermal desorption is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatcauses the sample to transfer into the gas phase. The gas phase sampleis then drawn into the mass spectrometer.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of less than 20% and an accuracy of 85% to 115%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the zero concentration.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a full scan mass spectrum showing possible C peptideprecursor ions. Details are discussed in Example 3.

FIG. 2 shows an exemplary fragmentation spectra (product ion scan) forfragmentation of a C peptide precursor ion with a m/z of about1007.5±0.50 across the m/z range of about 50 to 1200. Details arediscussed in Example 3.

FIG. 3 shows a plot of the linearity of quantitation of C peptide inspiked mock serum standards measured with MS/MS. Details are describedin Example 4.

FIG. 4 shows a plot of the linearity of quantitation of C peptide inspiked stripped serum samples measured with MS/MS. Details are describedin Example 4.

FIGS. 5A-C show mass spectra for the ionization of C peptide and itssodium adducts collected by scanning a high resolution/high accuracymass spectrometer across the m/z range of about 500 to 2000, 1005-1040,and 1519-1526, respectively. Details are discussed in Example 5.

FIG. 6 shows a mass spectra for the C peptide ion with a m/z of about1007.5±collected by scanning a high resolution/high accuracy massspectrometer across the m/z range of about 1005 to 1012. Details arediscussed in Example 5.

FIG. 7 shows a plot of the linearity of quantitation of C peptide inspiked mock serum standards measured with high resolution/high accuracyMS. Details are described in Example 6.

FIG. 8 shows a plot of the linearity of quantitation of C peptide inspiked stripped serum samples measured with high resolution/highaccuracy MS. Details are described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring the amount of C peptide in a sample.More specifically, mass spectrometric methods are described fordetecting and quantifying C peptide in a sample. The methods may utilizesolid phase extraction (SPE) and/or liquid chromatography (LC), toperform a purification of selected analytes, combined with methods ofmass spectrometry (MS), thereby providing an assay system for detectingand quantifying C peptide in a sample. The preferred embodiments areparticularly well suited for application in large clinical laboratoriesfor automated C peptide quantification assay.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as blood, plasma, serum, saliva, cerebrospinal fluid, ortissue samples; preferably plasma and serum. Such samples may beobtained, for example, from a patient; that is, a living person, male orfemale, presenting oneself in a clinical setting for diagnosis,prognosis, or treatment of a disease or condition. In embodiments wherethe sample comprises a biological sample, the methods may be used todetermine the amount of C peptide in the sample when the sample wasobtained from the biological source (i.e., the amount of endogenous Cpeptide in the sample).

The present invention also contemplates kits for a C peptidequantitation assay. A kit for a C peptide quantitation assay may includea kit comprising the compositions provided herein, such as an externalreference standard. The external reference standard, in some aspects,includes blank plasma or serum spiked with C peptide or an isotopicallylabeled variant thereof. For example, a kit may include packagingmaterial and measured amounts of an isotopically labeled internalstandard, in amounts sufficient for at least one assay. Typically, thekits will also include instructions recorded in a tangible form (e.g.,contained on paper or an electronic medium) for using the packagedreagents for use in a C peptide quantitation assay.

Calibration and QC pools for use in embodiments of the present inventionare preferably prepared using a matrix similar to the intended samplematrix, provided that C peptide is essentially absent.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, C peptide may beenriched relative to one or more other components in the sample byvarious methods known in the art, including for example, liquidchromatography, filtration, centrifugation, thin layer chromatography(TLC), electrophoresis including capillary electrophoresis, affinityseparations including immunoaffinity separations, extraction methodsincluding ethyl acetate or methanol extraction, and the use ofchaotropic agents or any combination of the above or the like.

One method of sample purification that may be used prior to massspectrometry is applying a sample to a solid-phase extraction (SPE)column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In this technique, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through.

In some embodiments, C peptide in a sample may be reversibly retained ona SPE column with a packing material comprising an alkyl bonded surface.For example, in some embodiments, a C-8 on-line SPE column (such as aStrata C-8 on-line SPE column (20 mm×2.0 mm) from Phenomenex, Inc. orequivalent) may be used to enrich C peptide prior to mass spectrometricanalysis. In some embodiments, use of an SPE column is conducted withHPLC Grade 0.1% aqueous formic acid as a wash solution, and use of 0.1%formic acid in acetonitrile as an elution solution.

In some embodiments, C peptide is not purified by any immunoaffinitytechnique. Some of these embodiments utilize a SPE column. In theseembodiments, the SPE column is not an immunoaffinity column.

In other embodiments, the methods include immunopurifying C peptideprior to mass spectrometry analysis. The immunopurification step may beperformed using any of the immunopurification methods well known in theart. Often the immunopurification procedure utilizes antibodies bound,conjugated, immobilized or otherwise attached to a solid support, forexample a column, well, tube, capsule, particle or the like. Generally,immunopurification methods involve (1) incubating a sample containingthe analyte of interest with antibodies such that the analyte binds tothe antibodies, (2) performing one or more washing steps, and (3)eluting the analyte from the antibodies.

In certain embodiments the incubation step of the immunopurification isperformed with the antibodies free in solution and the antibodies aresubsequently bound or attached to a solid surface prior to the washingsteps. In certain embodiments this can be achieved using a primaryantibody that is an anti-C peptide antibody and a secondary antibodyattached to a solid surface that has an affinity to the primary anti-Cpeptide antibody. In alternative embodiments, the primary antibody isbound to the solid surface prior to the incubation step.

Appropriate solid supports include without limitation tubes, slides,columns, beads, capsules, particles, gels, and the like. In somepreferred embodiments, the solid support is a multi-well plate, such as,for example, a 96 well plate, a 384-well plate or the like. In someembodiments the solid support are sepharose or agarose beads or gels.There are numerous methods well known in the art by which antibodies(for example, an anti-C peptide antibody or a secondary antibody) may bebound, attached, immobilized or coupled to a solid support, e.g.,covalent or non-covalent linkages adsorption, affinity binding, ioniclinkages and the like. In some embodiments antibodies are coupled usingCNBr, for example the antibodies may be coupled to CNBr activatedsepharose. In other embodiments, the antibody is attached to the solidsupport through an antibody binding protein such as protein A, proteinG, protein A/G, or protein L.

The washing step of the immunopurification methods generally involvewashing the solid support such that the C peptide remain bound to theanti-C peptide antibodies on the solid support. The elution step of theimmunopurification generally involves the addition of a solution thatdisrupts the binding of C peptide to the anti-C peptide antibodies.Exemplary elution solutions include organic solutions, salt solutions,and high or low pH solutions.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). In liquid chromatographytechniques, an analyte may be purified by applying a sample to achromatographic analytical column under mobile phase conditions wherethe analyte of interest elutes at a differential rate in comparison toone or more other materials. Such procedures may enrich the amount ofone or more analytes of interest relative to one or more othercomponents of the sample.

Certain methods of liquid chromatography, including HPLC, rely onrelatively slow, laminar flow technology. Traditional HPLC analysisrelies on column packing in which laminar flow of the sample through thecolumn is the basis for separation of the analyte of interest from thesample. The skilled artisan will understand that separation in suchcolumns is a partition process and may select LC, including HPLC,instruments and columns that are suitable for use with C peptide. Thechromatographic analytical column typically includes a medium (i.e., apacking material) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlestypically include a bonded surface that interacts with the variouschemical moieties to facilitate separation of the chemical moieties. Onesuitable bonded surface is a hydrophobic bonded surface such as an alkylbonded or a cyano bonded surface. Alkyl bonded surfaces may include C-4,C-8, C-12, or C-18 bonded alkyl groups. In some embodiments, thechromatographic analytical column is a monolithic C-18 column. Thechromatographic analytical column includes an inlet port for receiving asample and an outlet port for discharging an effluent that includes thefractionated sample. The sample may be supplied to the inlet portdirectly, or from a SPE column, such as an on-line SPE column or a TFLCcolumn. In some embodiments, an on-line filter may be used ahead of theSPE column and or HPLC column to remove particulates and phospholipidsin the samples prior to the samples reaching the SPE and/or TFLC and/orHPLC columns.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytypic (i.e.mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In some embodiments, C peptide in a sample is enriched with HPLC. ThisHPLC may be 1-D HPLC conducted with a monolithic C-18 columnchromatographic system, for example, an Onyx Monolithic C-18 column fromPhenomenex Inc. (50×2.0 mm), or equivalent. In certain embodiments, HPLCis performed using HPLC Grade 0.1% aqueous formic acid as a washsolution, and, and 0.1% formic acid in acetonitrile as an elutionsolution.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, one or more of the above purification techniquesmay be used in parallel for purification of C peptide to allow forsimultaneous processing of multiple samples. In some embodiments, thepurification techniques employed exclude immunopurification techniques,such as immunoaffinity chromatography.

In some embodiments, TFLC may be used for purification of C peptideprior to mass spectrometry. In such embodiments, samples may beextracted using a TFLC column which captures the analyte. The analyte isthen eluted and transferred on-line to an analytical HPLC column. Forexample, sample extraction may be accomplished with a TFLC extractioncartridge with a large particle size (50 μm) packing. Sample eluted offof this column may then be transferred on-line to an HPLC analyticalcolumn for further purification prior to mass spectrometry. Because thesteps involved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

Detection and Quantitation of C Peptide by Mass Spectrometry

Mass spectrometry is performed using a mass spectrometer, which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. In various embodiments, C peptide may beionized by any method known to the skilled artisan. For exampleionization of C peptide may be performed by electron ionization,chemical ionization, electrospray ionization (ESI), photon ionization,atmospheric pressure chemical ionization (APCI), photoionization,atmospheric pressure photoionization (APPI), Laser diode thermaldesorption (LDTD), fast atom bombardment (FAB), liquid secondaryionization (LSI), matrix assisted laser desorption ionization (MALDI),field ionization, field desorption, thermospray/plasmaspray ionization,surface enhanced laser desorption ionization (SELDI), inductivelycoupled plasma (ICP) and particle beam ionization. The skilled artisanwill understand that the choice of ionization method may be determinedbased on the analyte to be measured, type of sample, the type ofdetector, the choice of positive versus negative mode, etc. C peptidemay be ionized in positive or negative mode. In preferred embodiments, Cpeptide is ionized by ESI in positive ion mode.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass-to-charge ratio (m/z). Various analyzersfor determining m/z include quadrupole analyzers, ion traps analyzers,time-of-flight analyzers, Fourier transform ion cyclotron resonance massanalyzers, and orbitrap analyzers. Some exemplary ion trap methods aredescribed in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000,14:967-73.

The ions may be detected using several detection modes. For example,selected ions may be detected, i.e. using a selective ion monitoringmode (SIM), or alternatively, mass transitions resulting from collisioninduced dissociation or neutral loss may be monitored, e.g., multiplereaction monitoring (MRM) or selected reaction monitoring (SRM). In someembodiments, the mass-to-charge ratio is determined using a quadrupoleanalyzer. In a “quadrupole” or “quadrupole ion trap” instrument, ions inan oscillating radio frequency field experience a force proportional tothe DC potential applied between electrodes, the amplitude of the RFsignal, and the mass/charge ratio. The voltage and amplitude may beselected so that only ions having a particular mass/charge ratio travelthe length of the quadrupole, while all other ions are deflected. Thus,quadrupole instruments may act as both a “mass filter” and as a “massdetector” for the ions injected into the instrument.

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks, forfragment ion(s) and/or precursor ions are measured to determine theamount of C peptide. The relative abundance of a given ion may beconverted into an absolute amount of the original analyte usingcalibration standard curves based on peaks of one or more ions of aninternal or external molecular standard.

One may enhance the resolution of MS techniques employing certain massspectrometric analyzers through “tandem mass spectrometry,” or “MS/MS”.In this technique, a precursor ion (also called a parent ion) generatedfrom a molecule of interest can be filtered in an MS instrument, and theprecursor ion subsequently fragmented to yield one or more fragment ions(also called daughter ions or product ions) that are then analyzed in asecond MS procedure. By careful selection of precursor ions, only ionsproduced by certain analytes are passed to the fragmentation chamber,where collisions with atoms of an inert gas produce the fragment ions.Because both the precursor and fragment ions are produced in areproducible fashion under a given set of ionization/fragmentationconditions, the MS/MS technique may provide an extremely powerfulanalytical tool. For example, the combination offiltration/fragmentation may be used to eliminate interferingsubstances, and may be particularly useful in complex samples, such asbiological samples. In certain embodiments, a mass spectrometricinstrument with multiple quadrupole analyzers (such as a triplequadrupole instrument) is employed to conduct tandem mass spectrometricanalysis.

In certain embodiments using a MS/MS technique, precursor ions areisolated for further fragmentation and collision activated dissociation(CAD) is used to generate fragment ions from the precursor ions forfurther detection. In CAD, precursor ions gain energy through collisionswith an inert gas, and subsequently fragment by a process referred to as“unimolecular decomposition.” Sufficient energy must be deposited in theprecursor ion so that certain bonds within the ion can be broken due toincreased vibrational energy.

In some embodiments, C peptide in a sample is detected and/or quantifiedusing MS/MS as follows. C peptide is enriched in a sample by firstsubjecting the sample to SPE, then to liquid chromatography, preferablyHPLC, such as 1-D HPLC; the flow of liquid solvent from achromatographic analytical column enters the heated nebulizer interfaceof an MS/MS analyzer; and the solvent/analyte mixture is converted tovapor in the heated charged tubing of the interface. During theseprocesses, the analyte (i.e., C peptide) is ionized. The ions, e.g.precursor ions, pass through the orifice of the instrument and enter thefirst quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters,allowing selection of ions (i.e., selection of “precursor” and“fragment” ions in Q1 and Q3, respectively) based on their mass tocharge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ionsare fragmented. The first quadrupole of the mass spectrometer (Q1)selects for molecules with the m/z of a C peptide precursor ion.Precursor ions with the correct m/z are allowed to pass into thecollision chamber (Q2), while unwanted ions with any other m/z collidewith the sides of the quadrupole and are eliminated. Precursor ionsentering Q2 collide with neutral gas molecules (such as Argon molecules)and fragment. The fragment ions generated are passed into quadrupole 3(Q3), where the C peptide fragment ions are selected while other ionsare eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; in some embodiments the MS/MS is performed in positive ionmode. In certain embodiments, Q1 selects for precursor ions with an m/zof about 1007.5±0.5. In related embodiments, Q3 may select fragment ionswith m/z of about 927.6±0.5, and/or 785.4±0.5, and/or 646.1±0.5. Incertain embodiments, the relative abundance of a single fragment ion maybe measured. Alternatively, the relative abundances of two or morefragment ions may be measured. In these embodiments, the relativeabundances of each fragment ion may be summed to quantitatively assess Cpeptide originally in the sample.

Alternate modes of operating a tandem mass spectrometric instrument thatmay be used in certain embodiments include product ion scanning andprecursor ion scanning. For a description of these modes of operation,see, e.g., E. Michael Thurman, et al., Chromatographic-MassSpectrometric Food Analysis for Trace Determination of PesticideResidues, Chapter 8 (Amadeo R. Fernandez-Alba, ed., Elsevier 2005)(387).

In other embodiments, a high resolution/high accuracy mass analyzer maybe used for quantitative analysis of C peptide according to methods ofthe present invention. To obtain acceptable level of quantitativeresults, the mass spectrometer must be capable of exhibiting a resolvingpower (FWHM) of 10,000 or higher, with accuracy of about 50 ppm or lessfor the ions of interest; preferably the mass spectrometer exhibits aresolving power (FWHM) of 18,000 or higher and accuracy of about 5 ppmor less; such as a resolving power (FWHM) of 20,000 or higher andaccuracy of about 3 ppm or less; such as a resolving power (FWHM) of25,000 or higher and accuracy of about 3 ppm or less. Three exemplaryanalyzers capable of exhibiting the requisite level of performance for Cpeptide ions are orbitrap mass analyzers, certain TOF mass analyzers,and Fourier transform ion cyclotron resonance mass analyzers.

Elements found in biological active molecules, such as carbon, oxygen,and nitrogen, naturally exist in a number of different isotopic forms.For example, most carbon is present as ¹²C, but approximately 1% of allnaturally occurring carbon is present as ¹³C. Thus, some fraction ofnaturally occurring molecules containing at least one carbon atom willcontain at least one ¹³C atom. Inclusion of naturally occurringelemental isotopes in molecules gives rise to multiple molecularisotopic forms. The difference in masses of molecular isotopic forms isat least 1 atomic mass unit (amu). This is because elemental isotopesdiffer by at least one neutron (mass of one neutron ≈1 amu). Whenmolecular isotopic forms are ionized to multiply charged states, themass distinction between the isotopic forms can become difficult todiscern because mass spectrometric detection is based on the mass tocharge ratio (m/z). For example, two isotopic forms differing in mass by1 amu that are both ionized to a 5+ state will exhibit differences intheir m/z of only 0.2. High resolution/high accuracy mass spectrometersare capable of discerning between isotopic forms of highly multiplycharged ions (such as ions with charges of ±2, ±3, ±4, ±5, or higher).

Due to naturally occurring elemental isotopes, multiple isotopic formstypically exist for every molecular ion (each of which may give rise toa separately detectable spectrometric peak if analyzed with a sensitiveenough mass spectrometric instrument). The m/z ratios and relativeabundances of multiple isotopic forms collectively comprise an isotopicsignature for a molecular ion. In some embodiments, the m/z ratios andrelative abundances for two or more molecular isotopic forms may beutilized to confirm the identity of a molecular ion under investigation.In some embodiments, the mass spectrometric peak from one or moreisotopic forms is used to quantitate a molecular ion. In some relatedembodiments, a single mass spectrometric peak from one isotopic form isused to quantitate a molecular ion. In other related embodiments, aplurality of isotopic peaks are used to quantitate a molecular ion. Inthese later embodiments, the plurality of isotopic peaks may be subjectto any appropriate mathematical treatment. Several mathematicaltreatments are known in the art and include, but are not limited tosumming the area under multiple peaks, or averaging the response frommultiple peaks. An exemplary spectra demonstrating such a multipleisotopic forms of C peptide ions within a m/z range of about 1007.5 isseen in FIG. 6 . As seen in the exemplary spectra, peaks from variousisotopic forms are seen at 1007.1750, 1007.5092, 1007.8362, 1008.1745,1008.5081, 1008.8355. Note, however, that the precise masses observedfor isotopic variants of any ion may vary slightly because ofinstrumental variance.

In some embodiments, the relative abundance of one or more ion ismeasured with a high resolution/high accuracy mass spectrometer in orderto qualitatively assess the amount of C-peptide in the sample. In someembodiments, the one or more ions measured by high resolution/highaccuracy mass spectrometry are multiply charged C peptide ions. Thesemultiply charged ions may include one or more of ions with a m/z ofabout 1510.3 (2+ ion) and about 1007.3 (3+ ion).

Use of high resolution orbitrap analyzers has been reported forqualitative and quantitative analyses of various analytes. See, e.g.,U.S. Patent Application Pub. No. 2008/0118932 (filed Nov. 9, 2007);Bredehöft, et al., Rapid Commun. Mass Spectrom., 2008, 22:477-485; LeBreton, et al., Rapid Commun. Mass Spectrom., 2008, 22:3130-36; Thevis,et al., Mass Spectrom. Reviews, 2008, 27:35-50; Thomas, et al., J. MassSpectrom., 2008, 43:908-15; Schenk, et al., BMC Medical Genomics, 2008,1:41; and Olsen, et al., Nature Methods, 2007, 4:709-12.

The results of an analyte assay may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of C peptide.Methods of generating and using such standard curves are well known inthe art and one of ordinary skill is capable of selecting an appropriateinternal standard. For example, in preferred embodiments one or moreforms of isotopically labeled C peptide may be used as internalstandards. Numerous other methods for relating the amount of an ion tothe amount of the original molecule will be well known to those ofordinary skill in the art.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(²H), ¹³C, and ¹⁵N. One or more isotopic labels can be incorporated atone or more positions in the molecule and one or more kinds of isotopiclabels can be used on the same isotopically labeled molecule.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1: Sample Preparation

Mock serum samples containing various amounts of human C peptide wereprepared by spiking human C peptide in mock serum (40 mg/mL Bovine SerumAlbumin (BSA) in Phosphate Buffered Saline (PBS) buffer with 0.002%protease inhibitor AEBSF) at various concentrations for assessment oflinear response (discussed below in Example 4).

Human C peptide was also spiked in double charcoal stripped serumobtained from Golden West Biologicals, Inc. at various concentrations toassess linearity of response (discussed below in Example 4).

Example 2: Enrichment of C Peptide Prior to Mass Spectrometry

Sample injection of the above prepared human C peptide-spiked mock andstripped sera was performed with a Cohesive Technologies Aria TX-420system using Aria OS V 1.6 or newer software.

50 μL samples were introduced into a Strata C-8 on-line SPE column (20mm×2.0 mm) from Phenomenex, Inc. or equivalent) on-line solid phaseextraction column. The solid phase extraction column retained C peptidewhile letting other serum proteins and large molecules flow through.

C peptide was eluted off the extraction column with 0.1% formic acid in40% acetonitrile and onto the analytical column (Onyx monolithic C18analytical column from Phenomenex Inc. (50×2.0 mm). An HPLC gradient wasapplied to the analytical column, to separate C peptide from otheranalytes contained in the sample. Mobile phase A was 0.1% formic acid inwater and mobile phase B was 0.1% formic acid in acetonitrile. The HPLCgradient started with a 24.0% organic gradient which was ramped to 35.5%in approximately 90 seconds.

The C peptide enriched samples were then subjected to MS/MS or highresolution/high accuracy MS or MS/MS for quantitation of C peptide.

Example 3: Detection and Quantitation of C Peptide by Tandem MS

MS/MS was performed using a Thermo TSQ Vantage MS/MS system (ThermoElectron Corporation). The following software programs, all from ThermoElectron, were used in the Examples described herein: TSQ Ultra QuantumV 1.4.1 or newer, Xcalibur V 2.0 or newer, and LCQuan V 2.5 or newer.Liquid solvent/analyte exiting the analytical column flowed to theheated nebulizer interface of the MS/MS analyzer. The solvent/analytemixture was converted to vapor in the heated tubing of the interface.Analytes were ionized by ESI.

Ions passed to the first quadrupole (Q1). Several possible C peptideprecursor ions were observed at Q1 as peaks of 1007.5, 1510.38. Anexemplary Q1 spectra is seen in FIG. 1 . A triply charged C peptideprecursor ion with a m/z of 1007.5±0.50 was selected for fragmentation.Ions entering quadrupole 2 (Q2) collided with argon gas (at a collisioncell energy of 20 V) to generate ion fragments, which were passed toquadrupole 3 (Q3) for further selection. An exemplary fragmentationspectra collected from a Q3 scan (product ion scan) is shown in FIG. 2 .The following mass transitions were observed for fragmentation of the1007.5±0.50 precursor ion.

TABLE 1 Mass Transitions Observed for C Peptide (Positive Polarity)Precursor Ion Product Ions Analyte (m/z) (m/z) C peptide 1007.5 ± 0.50927.6 ± 0.50, 785.4 ± 0.50, 646.1 ± 0.50

Of the observed transitions, three were monitored in MRM mode and summedfor quantitative analysis: the precursor ion of 1007.5±0.50 to927.6±0.50, 785.4±0.50, and 646.1±0.50. Although quantitation wasaccomplished by monitoring three mass transitions, quantitation may beaccomplished by monitoring as few as a single mass transition.Conversely, additional mass transitions may be selected to replace oraugment, in any combination, any of the above monitored transitions.

Example 4: Tandem MS Data Analysis for Quantitation of C Peptide

C peptide quantitation via monitoring the indicated transitions with atriple quadrupole tandem mass spectrometer was conducted on C peptidespiked mock serum samples and spiked stripped serum samples.

To establish the linearity of C peptide detection in the assay, severalspiked mock serum standards and spiked stripped serum samples wereanalyzed across a concentration range of about 1 ng/mL to about 500ng/mL. Graphs showing the linearity of the data for C peptide detectionin spiked mock serum standards and spiked stripped serum samples areshown in FIGS. 3 and 4 , respectively. The goodness of fit (R²) for Cpeptide was determined to be 0.998 in mock serum, and 0.996 in strippedserum.

Example 5: Detection of C Peptide by High Resolution/High Accuracy MS

High resolution/high accuracy MS was performed using an Agilent TOF MSsystem (Agilent Technologies, Inc.). This system employs an QTOF MSanalyzer capable of high resolution/high accuracy MS. The instrumentexhibits resolution of approximately 10,000 FWHM, and mass accuracy ofapproximately 50 ppm while measuring C peptide.

Ionization is conducted with an ESI source in positive ion mode.Multiply charged C peptide ions were observed with m/z of 1510.3±0.50(for the 2+ ion) and 1007.5±0.50 (for the 3+ ion). An exemplary highresolution/high accuracy spectra across the range of about 500 to 2000,1005-1040, and 1519-1526, m/z showing C peptide ions is seen in FIGS.5A-5C respectively.

Data was collected for the ion with m/z of 1007.5±0.50 for quantitationof C peptide. A high resolution scan of this ion was collected and usedto confirm the relative abundances of the predicted natural isotopicdistribution. An exemplary high resolution/high accuracy spectra acrossthe range of about 1005 to 1012 is shown in FIG. 6 .

Example 6: High Resolution/High Accuracy MS Data Analysis forQuantitation of C Peptide

C peptide quantitation via monitoring the indicated transitions with ahigh resolution/high accuracy mass spectrometer was conducted on Cpeptide spiked mock serum samples and spiked stripped serum samples.

To establish the linearity of C peptide detection in the assay, severalspiked mock serum standards and spiked stripped serum samples wereanalyzed across concentration ranges of about 3.9 ng/mL to about 500ng/mL (spiked mock serum) and about 31.25 ng/mL to about 500 ng/mL(spiked stripped serum). Graphs showing the linearity of the data for Cpeptide detection in spiked mock serum standards and spiked strippedserum samples are shown in FIGS. 7 and 8 , respectively. The goodness offit (R 2) for C peptide was determined to be 0.998 in spiked mock serum,and 0.996 in spiked stripped serum.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed is:
 1. A method for determining the amount of C peptidein a sample by tandem mass spectrometry, the method comprising:purifying a sample containing C peptide by high performance liquidchromatography (HPLC); ionizing C peptide by in positive mode togenerate one or more C peptide ions detectable by mass spectrometry;determining the amount of one or more C peptide ions by tandem massspectrometry; wherein the amount of ions determined in step (c) isrelated to the amount of a C peptide in said sample.
 2. The method ofclaim 1, wherein said HPLC is 1-D HPLC.
 3. The method of claim 1,wherein further comprising subjecting the sample containing C peptide tosolid phase extraction (SPE).
 4. The method of claim 3, wherein said SPEand HPLC are conducted with on-line processing.
 5. The method of claim1, wherein said sample is from a human.
 6. The method of claim 1,wherein said sample is plasma.
 7. The method of claim 1, wherein saidsample is serum.
 8. The method of claim 1, wherein said massspectrometry is high resolution/high accuracy mass spectrometry.
 9. Themethod of claim 8, wherein said high resolution/high accuracy massspectrometry is conducted at a FWHM of 10,000 and a mass accuracy of 50ppm.
 10. The method of claim 8, wherein said high resolution/highaccuracy mass spectrometer is a high resolution/high accuracytime-of-flight (TOF) mass spectrometer.
 11. The method of claim 1,wherein the one or more C peptide ions comprise a fragment ion having amass to charge ratio of 646.1±0.5.